Aetas Amponnawarat
| Aetas Amponnawarat |
Amponnawarat, Aetas, Chompunud Na Ayudhya, Chalatip, Ali, Hydar
University of Pennsylvania School of Dental Medicine, Department of Basic and Translational Sciences
Pseudomonas aeruginosa is a common cause of nosocomial skin wound infection and presents a rising therapeutic challenge due to its ability to form biofilms and increase antibiotic resistance. In mouse skin, mast cells (MCs) contribute to both bacterial clearance and wound healing following P. aeruginosa infection model via an unknown mechanism. We have previously shown that host defense peptides (HDPs) activate human MCs via Mas-related G protein-coupled receptor X2 (MRGPRX2). Small molecule HDP mimetics are relatively inexpensive to synthesize and have distinct advantages over HDPs in terms of stability, bioavailability, and low toxicity. Murepavadin is a lipidated protegrin-1 mimetic which has specific activity against a broad panel of P. aeruginosa including multidrug-resistant clinical isolates. Given that protegrin-1 activates human MCs via MRGPRX2, this raises an interesting possibility that potential therapeutic utility of murepavadin for P. aeruginosa skin infection likely reflects the activation of MCs via MRGPRX2, in addition to its direct antimicrobial activity.
MethodsCa2+ mobilization and degranulation assays were used to determine murepavadin-mediated MC activation in a human MC line (LAD2) endogenously expresses MRGPRX2 and RBL-2H3 cells stably expressing MRGPRX2 (RBL-MRGPRX2). Chemokine/cytokine generation following MC activation was measured by ELISA. Site-directed mutagenesis was used to construct missense MRGPRX2 variants. A transcriptional activation following arrestin translocation (Tango) assay in engineered HEK293 cells was used for -arrestin recruitment. Cell surface receptor expression and internalization following activation by murepavadin were determined by flow cytometry. Furthermore, murepavadin-induced degranulation in vivo was measured by Evan’s Blue extravasation.
ResultsWe found that murepavadin induced Ca2+ mobilization, degranulation, chemokine IL-8 and CCL3 production in LAD2 cells. Murepavadin also induced degranulation in RBL-2H3 expressing MRGPRX2 but this response was significantly inhibited in cells expressing missense MRGPRX2 variants. Compound 48/80 induced β‐arrestin recruitment as measured by Tango and promoted receptor internalization, which resulted in substantial decrease in the subsequent responsiveness to the MRGPRX2 agonist. By contrast, murepavadin did not cause β‐arrestin‐mediated MRGPRX2 downregulation. Murepavadin induced degranulation in mouse peritoneal MCs via MrgprB2 (ortholog of human MRGPRX2) and caused increased vascular permeability in wild-type mice but this response was abolished in MrgprB2-/- mice. These findings demonstrate that murepavadin activates human and murine MCs via MRGPRX2 and MrgprB2, respectively and that MRGPRX2 is resistant to β‐arrestin‐mediated downregulation.
ConclusionMurepavadin activates MCs via MRGPRX2 and MrgprB2